Intestinal carriage of multidrug-resistant bacteria among healthcare professionals in Germany

Healthcare professionals (HCP) might be at increased risk of acquisition of multidrug-resistant bacteria (MDRB), i.e., methillicin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and multidrug-resistant gram-negative bacteria (MDRGN) and could be an unidentified source of MDRB transmission. The aim of this study was to determine the prevalence as well as risk factors of MDRB colonization among HCP. HCP (n=107) taking part in an antibiotic stewardship program, were voluntarily recruited to perform a rectal swab and to fill in a questionnaire to identify risk factors of MDRB carriage, i.e. being physician, gender, travel abroad within the previous 12 months, vegetarianism, regular consumption of raw meat, contact to domestic animals, household members with contact to livestock, work or fellowship abroad, as well as medical treatment abroad and antibiotic therapy within the previous 12 months. Selective solid media were used to determine the colonization rate with MRSA, VRE and MDRGN. MDRGN were further characterized by molecular analysis of underlying β-lactamases. None of the participants had an intestinal colonization with MRSA or VRE. 3.7% of the participants were colonized with extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, predominantly blaCTX-M type. Neither additional flouroquinolone resistance nor carbapenem resistance was detected in any of these isolates. No risk factors were identified to have a significant impact of MDRB carriage among HCP. A colonization rate of 3.7% with ESBL-producing Enterobacteriaceae is of interest, but comparing it to previously published data with similar colonization rates in the healthy population in the same geographic area, it is probably less an occupational risk.

A study reports that about 20% of high risk patients, i.e. patients from nursing homes, have an intestinal colonization with S. aureus and about 9% of the patients are colonized with MRSA [4]. Nasal carriage seems to be a predisposition for intestinal carriage [4]. MRSA carrier can be categorized into transient and persistent carriers. Persistent carriers are described to be colonized on several sites over months or years. They are more likely to have endogenous infections due to their MRSA strain compared to transient carriers [5], [6]. Persistent carriers are defined by remaining colonized unless they are treated with an effective agent, in comparison to transient carriers, who are described to clean their colonization even in the absence of an effective treatment [5]. Transmission frequently results from the transient colonization through the hands of hospital staff, carrying strains from one to the other patient [7]. VRE is the third most common nosocomial agent in the German healthcare setting, showing an increased prevalence from 9.3% in 2008 to 18.5% in 2012 in hospital settings [8]. The number of nosocomial infections with VRE increased from 3.9% in 2007/2008 to 7.2% in 2013/2014 [9]. In the neighbouring countries of Germany, the prevalence of VRE has been described to be lower, e.g. Denmark 2.0%, the Netherlands 0.0%, France 0.8%, Belgium 1.4%, Austria 3.2% [8].
MDRGN and in particular ESBL-producing Enterobacteriaceae are reported to colonize patients at risk as well as healthy people with increasing frequency [10]. The introduction of third generation cephalosporins in the 1980s [11] was a milestone in the treatment of gram-negative infections. This increase of selective pressure might also have promoted the spread of resistant organisms throughout Europe. The first cases of extended-spectrum β-lactamase producing Klebsiella pneumoniae and Serratia marcescens isolates were published in Germany in 1983 [12], [13]. According to EARS-data, the percentage of invasive E. coli isolates resistant to third generation cephalosporins increased from 0% in year 2000 to 10.4% in year 2015 in Germany [14]. A study conducted on the prevalence of fecal carriage in the healthy population in Germany between October 2009 and November 2012, demonstrated a frequency of 6.3% ESBL-producing E. coli, predominantly (95,1%) bla CTX-M type [15]. The aim of this study was to investigate the prevalence of intestinal MDRB colonization in healthcare professionals (HCP) and to identify potential risk factors of MDRB carriage.

Materials and methods
HCP, who took part in the antibiotic stewardship program supported by the German Society for Infectious Diseases, the divisions of Infectious Diseases Freiburg and Dresden, were recruited from March 2013 till March 2014. Participation was voluntary and anonymous. Volunteers were asked to perform a rectal swab and to fill out a questionnaire. This questionnaire addressed the potential risk factors of MDRB carriage, i.e. being physician, gender, travel abroad, vegetarianism, consumption of raw meat, contact to domestic animals, household members with contact to livestock, work or fellowship abroad, as well as medical treatment abroad and antibiotic therapy within the previous 12 months.

Characterization of MDRGN
MDRGN is defined as Enterobacteriaceae with extended spectrum beta-lactamase (ESBL)-phenotype as well as Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii resistant against at least piperacillin, any 3 rd /4 th generation cephalosporin and ciprofloxacin [16], [17]. ESBL phenotype was identified by VITEK 2 Advanced Expert System™ (bioMérieux). ESBL encoding bla genes, i.e. bla CTX-M , bla TEM and bla SHV , were detected via PCR and subtype analysis was done by subsequent sequencing, as published by Dallenne C. et al. [18].

Statistical analysis
Categorical data i.e. being physician, gender, travel abroad, vegetarianism, consumption of raw meat, contact to livestock and contact to domestic animals, work or fellowship abroad, as well as medical treatment abroad and antibiotic therapy within the previous 12 months, were analyzed using Fisher's exact test with the exception for datasets where a cell equaled zero. In these cases, the Pearson, Mantel and Haenszel chi-squared test was applied using a continuity correction. Odds ratios (OR) and 95% confidence intervals (CI) are given. All tests were performed two-tailed, and a p-value ≤0.05 was considered as statistically significant.

Ethics
After consultation with the head of the ethic committee at the Goethe University Hospital Frankfurt no further approval for this survey was deemed necessary.

Discussion
Antibiotic resistance is recognized as a major threat to modern medicine [19]. Patients infected by a MDRB have a higher mortality and an increased morbidity, leading to a boost of costs [1], [2], [3]. Since intestinal colonization with MDRB is one of the most frequent reservoirs of infections [20], we intended to investigate the prevalence of intestinal colonization with MDRB in HCP. Fecal carriage of MDRB in the population has previously been described in several studies, e.g. by Valenza et al. in 2014 [15] reporting 6.3% of ESBL-producing Enterobacteriaceae colonization in Germany. Furthermore Meyer et al. found a rate of 3.5% ESBL-producing Enterobacteriaceae carriage among healthy infection control personnel in 2011 [21]. In our setting, we found a prevalence of 3.7% ESBL carriage among HCP, which is consistent with the published data [15], [21]. Molecular characterization of ESBL-encoding genes revealed the presence of bla CTX-M-1 and bla CTX-M-14 . This result reflects the published predominance of these genes together with bla CTX-M-15 in Germany [22] and also is in accordance with epidemiological studies that reported the shift from TEM and SHV enzymes being highly prevalent until the 1990s to CTX-M group enzymes in Europe [15], [22], [23], [24], [25]. The prevalence of ESBL-producing Enterobacteriaceae geographically varies. Geser et al. found that 5.8% staff members of a meat-processing company in Switzerland were colonized with ESBL [24]. A Swedish study showed a prevalence of ESBL-producers of 2.9% among healthy preschool children in 2010 [26]. Data from France showed a prevalence of ESBL-producing Enterobacteriaceae carriers in 2009 and 2011 of 2.1% and 6%, respectively [27], [28]. The prevalence of ESBL-producing Enterobacteriaceae carriage is much higher in Asia than Europe. Li et al. found a prevalence of ESBL-producing E. coli of 50.5% in healthy individuals in China in 2011 [25], predominantly harbouring CTX-M enzymes. Another study from Thailand observed a prevalence of 61.7% Enterobacteriaceae showing an ESBL phenotype and 58.2% out of total 160 participants had a CTX-M enzyme [29]. Several studies [30], [31] investigated the acquisition of ESBL-producing Enterobacteriaceae among participants during travel. Post-travel analysis demonstrated that 35% of the participants were colonized with a new ESBL strain during travel, mostly acquired in South-East Asia region [30]. In this study, no significant risk factor associated with the carriage of ESBL-producing Enterobacteriaceae was identified. Though some participants matched risk factors which have previously been described in the literature, i.e. travel [30], [31], companion animals or livestock contact [21]. In our study, neither travel history nor work or fellowship abroad in the previous 12 months could be described as a significant risk factor for ESBL carriage. Limitation of our data is that we investigated only a small number of participants in a cohort of HCP who might be sensitized to hand hygiene and infection control measurements, and might therefore not be representative for all HCP.

Conclusion
In summary, we indentified a colonization rate of 3.7% of ESBL-producing Enterobacteriaceae in HCP. Since this colonization rate does not differ from the healthy population in this geographical area and no other MDRB have been identified in this cohort, HCP presumably do not have an occupational risk of MDRB colonization.

Competing interests
The authors declare that they have no competing interests.